Transgenic plants carrying neoxanthin cleavage enzyme gene

ABSTRACT

A DNA encoding a neoxanthin cleavage enzyme used for improving stress tolerance in a plant, a method for increasing stress tolerance in a plant by introducing the DNA into the plant, and a transgenic plant into which a neoxanthin cleavage enzyme gene is introduced, are provided. A DNA used for reducing stress tolerance in a plant, a method for decreasing stress tolerance in a plant by introducing the DNA into the plant, and a transgenic plant into which the DNA is introduced, are also provided. The present invention enables creating a plant in which stress tolerance has been increased or decreased.

FIELD OF THE INVENTION

The present invention relates to a DNA for improving or reducing stresstolerance in a plant, and a transgenic plant of the DNA.

BACKGROUND OF THE INVENTION

Plants must adapt themselves to various stresses, for example, drought,salt in the soil, and low temperature because they can not move freely.Among these stresses, drought is thought to effect plant growth the mostseverely. In order to survive in drought condition, some plants haveacquired a physiologically and/or morphologically specific trait in theevolutional process while many other plants also confer a mechanism toresponse to the drought stress and defend themselves. These responses toa shortage of water and adaptation to drought environment in plants arecaused by various physiological changes including the alternation ofgene expression at drought (Shinozaki, K and Yamaguchi-Shinozaki, K.,Plant Physiol., 115: 327–334, 1997; Shinozaki, K. andYamaguchi-Shinozaki, K., “Molecular responses to drought stress.” InShinozaki and Yamaguchi-Shinozaki (eds), “Molecular responses to cold,drought, heat and salt stress in higher plants,” R. G. LANDES company,Austin, Tex., USA, pp. 11–28, 1999). For example, in Arabidopsis(Arabidopsis thaliana), it is known that a drought signal is transmittedthrough an abscisic acid (ABA) dependent pathway and ABA independentpathway to control the gene expression involved in drought tolerance.These gene products are thought to have a function in controlling, forexample, accumulation of osmoprotectants such as sucrose and proline,half life of proteins, stress signal transduction pathway, andtranscription (Bray, E. A., Trends in Plant Science, 2: 48–54, 1997;Bohnert, H. J. et al., Plant Cell, 7: 1099–1111, 1995; Ingram, J. andBartels, D., Annu. Rev. Plant Physiol. Plant Mol. Biol., 47: 377–403,1996; Shinozaki, K. and Yamaguchi-Shinozaki, K., Plant Physiol., 115:327–334, 1997; Shinozaki, K. and Yamaguchi-Shinozaki, K., “Molecularresponses to drought stress.” In Shinozaki and Yamaguchi-Shinozaki(eds), “Molecular responses to cold, drought, heat and salt stress inhigher plants,” R. G. LANDES company, Austin, Tex., USA, pp. 11–28,1999).

C40 pathway has been proposed as a biosynthetic pathway of ABA in higherplants. The C40 pathway, also called a carotenoid pathway, is asynthetic pathway through epoxydation of zeaxanthin, synthesizingviolaxanthin, neoxanthin, xanthoxin, ABA aldehyde, and then ABA(Zeevaart, J. A. D. and Creelman R. A., Ann. Rev. Plant Physiol. PlantMol. Biol., 39: 439–473, 1988). This biosynthetic pathway has beenproposed from physiological studies and analyses of ABA biosyntheticvariants. For example, variant aba2 isolated from tobacco (Nicotianatabacum) has a mutation in a gene (aba2) of zeaxanthin epoxidase enzymewhich catalyzes the epoxidation of zeaxanthin (Marin E. et al., EMBO J.,15:2331–2342, 1996). Variant vp14 isolated from maize has a mutation ina gene (VP14) of neoxanthin cleavage enzyme which catalyzes theconversion from a neoxanthin to xanthoxin (Tan, B. C. et al., Proc.Natl. Acad. Sci. USA, 94: 12235–12240, 1997). From Arabidopsis plants,variant aba3 having a mutation in an enzyme which catalyzes a reactionfrom xanthoxin to ABA aldehyde, and variant aba4 involved in thereaction for oxidizing ABA aldehyde to produce ABA have been isolated(Schwartz, S. H. et al., Plant Physiol., 114: 161–166, 1997;Leon-Kloosterziel, K. M. et al., Plant J., 10: 655–661, 1996).

A maize having a mutation in a neoxanthin cleavage enzyme gene (VP14) isknown to show a trait of easily loosing water and easily wilting. It hasnot been known yet, however, whether stress tolerance in plants can beimproved or not using the neoxanthin cleavage enzyme gene.

SUMMARY OF THE INVENTION

An objective of the present invention is to provide a DNA encoding aneoxanthin cleavage enzyme used for improving stress tolerance in aplant, a method for increasing stress tolerance in a plant byintroducing the DNA into the plant, and a transgenic plant into which aneoxanthin cleavage enzyme gene is introduced. Another objective of thepresent invention is to provide a DNA used for reducing stress tolerancein a plant, a method for decreasing stress tolerance in a plant byintroducing the DNA into the plant, and a transgenic plant into whichthe DNA is introduced. The improvement of stress tolerance in plants isuseful, for example, in plant breeding.

The present inventors have isolated a cDNA clone (CPRD65) correspondingto a gene involved in a response against drought treatment, by thedifferential screening of a cDNA library prepared from a cowpea plant(Vigna unguiculata) which showed extensive drought tolerance afterdehydration treatment for 10 hours. The CPRD65 cDNA was expected toencode a neoxanthin cleavage enzyme proposed to be involved inbiosynthesis of abscisic acid (ABA). Drought stress given to an8-day-old cowpea plant strongly induced the accumulation of ABA and theexpression of the CPRD65, indicating the potential of the profoundinvolvement of CPRD65 gene, especially in the response to droughtstress. Determination of an enzyme activity using GST-CPRD65 fusionprotein confirmed that the CPRD65 comprises an activity of cleaving9-cis-neoxanthin to produce xanthoxin. These results indicate that theCPRD65 gene encodes a neoxanthin cleavage enzyme and its product plays akey role in endogenous ABA biosynthesis under drought stress.

Moreover, the present inventors have isolated a novel gene (AtNCED3) byscreening a neoxanthin cleavage enzyme gene from an Arabidopsisplant-derived cDNA library using a cDNA of the CPRD65 gene isolated fromcowpea plants as a probe. In addition, four types of sequences (AtNCED1,2, 4, and 5) derived from an Arabidopsis plant comprising high homologywith these genes were identified. Expression of these genes inEscherichia coli (E. coli) and assay of a neoxanthin cleavage activityrevealed that AtNCED1, 3, and 5 comprise a neoxanthin cleavage enzymeactivity same as the CPRD65.

The present inventors first produced a transgenic plant of Arabidopsisusing AtNCED3, a neoxanthin cleavage enzyme gene. The AtNCED3 gene wasligated downstream of 35S promoter in a vector for introducing a geneinto plant cells (pBE2113N) in the directions of sense (anoverexpression type) or antisense (an expression inhibition type) andintroduced the vector into Arabidopsis by the vacuum infiltrationmethod. Evaluation of drought tolerance of the prepared transgenicplants revealed that stress tolerance in the overexpressed plants wassignificantly increased compared with that in their parent lines. Incontrast, in the expression-inhibited lines into which the antisense wasintroduced, stress tolerance was reduced (FIGS. 15 and 16). In such amanner, the present inventors found that actually the transgenic plantinto which the neoxanthin cleavage enzyme gene is introducedsignificantly increased stress tolerance and stress tolerance can besignificantly reduced by decreasing the expression of the gene tocomplete the present invention.

Specifically, this invention relates to a DNA encoding a neoxanthincleavage enzyme used for improving stress tolerance in a plant, a methodfor increasing stress tolerance in a plant by introducing the DNA intothe plant, and a transgenic plant into which a neoxanthin cleavageenzyme gene is introduced, as well as a DNA used for reducing stresstolerance in a plant, a method for decreasing stress tolerance in aplant by introducing the DNA into the plant, and a transgenic plant intowhich the DNA is introduced, and more specifically, the presentinvention provides:

-   (1) an isolated DNA encoding a protein having a neoxanthin cleavage    activity for improving stress tolerance in a plant,-   (2) an isolated DNA for reducing stress tolerance in a plant,    wherein the DNA is selected from the group consisting of:-   (a) a DNA encoding an antisense RNA complementary to a transcript of    a gene encoding a protein having a neoxanthin cleavage activity;-   (b) a DNA encoding an RNA comprising a ribozyme activity which    cleaves a transcript of a gene encoding a protein having a    neoxanthin cleavage activity; and-   (c) a DNA encoding an RNA which inhibits the expression of a gene    encoding a protein having a neoxanthin cleavage in a plant cell by    the cosuppression effect,-   (3) the DNA of (1) or (2), wherein the protein having a neoxanthin    cleavage activity is selected from the group consisting of:-   (a) a protein comprising an amino acid sequence of SEQ ID NOs: 2    (AtNCED1), 6 (AtNCED3), 10 (AtNCED5), 12 (CPRD65), 14 (VP14), or 16    (LeNCED1),-   (b) a protein comprising an amino acid sequence in which one or more    amino acids in SEQ ID NOs: 2 (AtNCED1), 6 (AtNCED3), 10 (AtNCED5),    12(CPRD65), 14 (VP14), or 16 (LeNCED1) are replaced, deleted, added,    and/or inserted, and-   (c) a protein encoded by a DNA which hybridizes with a DNA    comprising a nucleotide sequence of SEQ ID NOs: 1 (AtNCED1), 5    (AtNCED3), 9 (AtNCED5), 11 (CPRD65), 13 (VP14), or 15 (LeNCED1)    under the stringent condition,-   (4) the DNA of any one of (1) to (3), wherein the protein having a    neoxanthin cleavage activity is derived from Arabidopsis plants,-   (5) a transformant plant cell carrying the DNA of any one of (1) to    (4),-   (6) a transgenic plant comprising the transformant plant cell of    (5),-   (7) a transgenic plant which is offspring or a clone of the    transgenic plant of (6),-   (8) the transgenic plant of (6) or (7), wherein the expression of a    gene encoding a protein having a neoxanthin cleavage activity is    increased or decreased compared with its wild type,-   (9) the transgenic plant of any one of (6) to (8), wherein the    amount of abscisic acid is increased or decreased compared with its    wild type,-   (10) the transgenic plant of any one of (6) to (9), wherein stress    tolerance is increased or decreased compared with its wild type,-   (11) a propagation material for the transgenic plant of any one    of (6) to (10),-   (12) a vector comprising the DNA of any one of (1) to (4),-   (13) a method for producing the transgenic plant of any one of (6)    to (10), comprising the steps of introducing the DNA of any one    of (1) to (4) into a plant cell and regenerating a plant from the    plant cell,-   (14) a method for increasing or decreasing stress tolerance in a    plant, comprising expressing the DNA of any one of (1) to (4) in a    plant cell,

In the present invention, “stress tolerance” means tolerance againstenvironmental stresses, for example, drought stress tolerance, saltstress tolerance, low temperature stress tolerance, air pollutiontolerance, tolerance to low oxygen condition, pathogen tolerance, drugtolerance such as those to agrochemicals, etc. Exogenous treatment withABA has known to improve tolerance against these stresses in many plants(refer to Takahashi, N. and Masuda, Y. (eds), “Plant Hormone Handbook(The Last),” Baifukan, Japan, pp. 78–160; and references cited therein).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Northern blot analysis of the expression of the CPRD65genes upon dehydration or rehydration. Total RNA was prepared from8-day-old cowpea plants that had been dehydrated for 0, 1, 2, 4, 6, 8,10, and 12 hours or rehydrated for 0, 1, 2, 5, 10, and 24 hours afterdehydration for 10 hours. Each lane was loaded with 10 μg of total RNA.The RNA was fractionated on a 1% agarose gel, blotted onto a nylonmembrane, and probed with [³²p]-labeled cDNA inserts of the CPRD65clones.

FIG. 2 shows comparison of the deduced amino acid sequences of theCPRD65, VP14 (neoxanthin cleavage enzyme from Zea mays, Schwartz, S. H.et al., Science, 276: 1872–1874, 1997), and LeNCED1 protein (neoxanthincleavage enzyme from Lycopersicon esculentum, Burbidge, A. etal., J.Exp. Bot., 47:2111–2112,1997; Burbidge, A. et al., Plant J., 17:427–431, 1999). Dashes indicate gaps that were introduced to optimizethe alignment. Enclosed boxes indicate identical amino acids. Shadowedregions indicate similar amino acids.

FIG. 3 shows Southern blot analysis of genomic DNA from cowpea 2246cultivar. Genomic DNA (10 μg per lane) was digested with EcoRI (E),HindIII (H), and XbaI (X), fractionated on a 1% agarose gel, andtransferred to a nylon membrane. The filter was allowed to hybridizewith a [³²P]-labeled fragment of the CPRD65 cDNA. “A” and “B” representdifferent stringency in hybridization conditions (refer to Examples).The size marker of DNA fragments is indicated in kbp.

FIG. 4(A) shows Northern blot analysis of the induction of the CPRD65gene by high salinity (NaCl), high temperature (heat), low temperature(cold), and the application of abscisic acid (ABA). Total RNA wasisolated from the cowpea plants at the indicated hours after thetreatment. Each lane was loaded with 10 μg of total RNA. The numberabove each lane indicates the duration (hours) of the treatment.

FIG. 4(B) shows Northern blot analysis of the CPRD65 gene without orwith 10 hour-dehydration treatment. Each lane was loaded with 10 μg oftotal RNA isolated from leaves (L), stems (S), and roots (R) of cowpea2246 cultivar. The RNA was fractionated on a 1% agarose gel, blottedonto a nylon membrane, and probed with [³²P]-labeled cDNA inserts of theCPRD65.

FIG. 5 shows HPLC profiles of carotenoid metabolites of GST (A) or theGST-CPRD65 recombinant protein (B). The reaction mixture containedcis-neoxanthin as a substrate. cN; cis-neoxanthin, C25; C25-product.

FIG. 6 shows plastid targeting of the CPRD65N-sGFP chimeric protein inprotoplasts. Constructs carrying the 35S-sGFP (A, C, E) or the35S-CPRD65N-sGFP chimeric constructs (B, D, F) were transfected into A.thaliana protoplasts using polyethylene glycol (PEG). Transfectedprotoplasts were observed by optical microscopy (A, B) or fluorescentmicroscopy with an interference filter type green (E, F) or red (C, D).E and F indicate GFP localization, and C and D chloroplast.

FIG. 7 shows the relationship between the accumulation of ABA and theexpression of the gene for CPRD65 during dehydration. The radioactivityretained on the nylon filter in FIG. 1 was quantified and plotted asshown. The procedure for quantification of ABA is described in Examples.Error bars show standard errors. The experiment was repeated threetimes.

FIG. 8 shows the accumulation of endogenous ABA in leaves (L), stems(S), and roots (R) of cowpea plants during dehydration treatment afterseparation of organs. The procedure for quantification of ABA is thesame as described in Example 7 (FIG. 7).

FIG. 9 shows comparison of the deduced amino acid sequences of AtNCED3and CPRD65. Dashes indicate gaps that were introduced to optimize thealignment. Enclosed boxes indicate identical amino acids. Shadowedregions indicate similar amino acids.

FIG. 10 shows alignments of amino acid sequences of AtNCED1,2,3,4, and5. Dashes indicate gaps that were introduced to optimize the alignment.Enclosed boxes indicate identical amino acids. Shadowed regions indicatesimilar amino acids.

FIG. 11 shows the result of the phylogenic analysis to examinerelationship between the amino acid sequences of AtNCED1, 2,3,4,5, andCPRD65, and their related sequences on the data bases. LeNCED1 (Ac. No.Z97215) and VP14 (Ac. No. U95953) show the proteins derived fromtomatoes and maize, respectively.

FIG. 12 shows the expression of the AtNCED genes against each stress.

FIG. 13 shows the expression of the AtNCED3 gene inAtNCED3-transformants. The upper and lower panels show the expression ofthe AtNCED3 gene in plants before drought and after drought stresstreatment, respectively. Two strains were used for both of plantsexpressing the sense AtNCED3 gene (overexpression) (A and B) and plantsexpressing the antisense (expression inhibition) (C and D).

FIG. 14 shows endogenous ABA amounts in AtNCED3-transformants.Endogenous ABA amounts increased in plants expressing the sense AtNCED3gene (overexpression) (A and B) but decreased in those expressing theantisense (expression inhibition) (C and D), compared with wild typeplants.

FIG. 15 shows the result of testing drought tolerance in neoxanthincleavage enzyme transgenic plants, indicating the plants and relativewater content in the leaves 14 days after the termination of theirrigation.

FIG. 16 shows the result of testing drought tolerance in neoxanthincleavage enzyme transgenic plants, indicating the plants 17 days afterthe termination of the irrigation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an isolated DNA encoding a proteinhaving a neoxanthin cleavage activity used for improving stresstolerance. A neoxanthin cleavage enzyme has been known as an enzymeinvolved in the ABA biosynthesis, however, has not been confirmedwhether introduction of the DNA encoding this enzyme into a plantactually leads to ABA accumulation and improvement of tolerance againststresses without a grave effect to plant's growth.

Exogenous treatment with ABA causes, for example, growth inhibition inmany plants. In a seed, it is known that ABA also causes growthinhibition (germination inhibition) (Takahashi, N. and Masuda, Y. (eds),“Plant Hormone Handbook (The Last),” Baifukan, Japan, pp. 78–160; andreferences cited therein). Increase in ABA level brings about variousdamages to plants. There has been no report whether excessive productionof ABA by an exogenous gene leads to acquirement of stress tolerance ornot. The conventional experimental procedures for exogenous treatmentwith ABA require the treatment at high concentration, which stronglyinhibits the growth and has prevented accurate evaluation of tolerance.Furthermore, experiments of exogenous treatments have not identifiedthat an appropriate level of ABA ensures normal growth and acquirementof tolerance. By obtaining ABA biosynthesis gene and creating atransgenic plant using this gene, the present inventors have firstconfirmed that stress tolerance in a plant can be improved.

An “isolated DNA” is a DNA the structure of which is not identical tothat of any naturally occurring DNA or to that of any fragment of anaturally occurring genomic DNA spanning more than three separate genes.The term therefore includes, for example, (a) a DNA which has thesequence of part of a naturally occurring genomic DNA molecule in thegenome of the organism in which it naturally occurs; (b) a DNAincorporated into a vector or into the genomic DNA of a prokaryote oreukaryote in a manner such that the resulting molecule is not identicalto any naturally occurring vector or genomic DNA; (c) a separatemolecule such as a cDNA, a genomic fragment, a fragment produced bypolymerase chain reaction (PCR), or a restriction fragment; and (d) arecombinant nucleotide sequence that is part of a hybrid gene, i.e., agene encoding a fusion protein. Specifically excluded from thisdefinition are DNA molecules present in mixtures of different (i) DNAmolecules, (ii) transfected cells, or (iii) cell clones; e.g., as theseoccur in a DNA library such as a cDNA or genomic DNA library.

As an isolated DNA used for increasing stress tolerance, any genes canbe used as long as it encodes a protein having a neoxanthin cleavageactivity. For example, VP14 of maize (Zea mays) (Schwartz, S. H. et al.,Science, 276:1872–1874, 1997; Tan, B. V. et al., Proc. Natl. Acad. Sci.USA, 94: 12235–12240, 1997) (cDNA: SEQ ID NO: 13, protein: SEQ ID NO:14), LeNCED1 of tomato (Lycopersicon esculentum) (Burbidge, A. et al.,J. Exp. Bot., 47: 2111–2112, 1997; Burbidge, A. et al., Plant J., 17:427–431, 1999) (cDNA: SEQ ID NO: 15, protein: SEQ ID NO: 16), and suchhave been isolated as neoxanthin cleavage enzyme genes. These genes areuseful for improving stress tolerance and can be used for the presentinvention. In addition, DNAs encoding AtNCED1 (SEQ ID NO: 2), AtNCED3(SEQ ID NO: 6), AtNCED5 (SEQ ID NO: 10), and CPRD65 (SEQ ID NO: 12) (SEQID NOs: 1, 5, 9, and 11, respectively) can be conveniently used.Moreover, a DNA encoding a neoxanthin cleavage enzyme of SEQ ID NO: 18(cDNA: SEQ ID NO: 17, protein: SEQ ID NO: 18) (Neill, S. J. et al., J.Exp. Bot., 49: 1893–1894, 1998, Ac. No. AJ005813) can also be used inthis invention. A DNA encoding a protein having a neoxanthin cleavageactivity can also be used as a reagent for increasing stress tolerance(stress tolerance increasing agent). The present invention also providesuses of a DNA encoding a protein having a neoxanthin cleavage activityfor increasing stress tolerance.

A DNA encoding a protein having a neoxanthin cleavage activity in thepresent invention includes a genomic DNA, a cDNA, and a chemosyntheticDNA. A genomic DNA and a cDNA can be prepared by common methods for oneskilled in the art. A genomic DNA can be prepared, for example, byextracting a genomic DNA from a plant according to conventional methods,in which a genomic library is prepared (in which, as a vector, forexample, a plasmid, a phage, a cosmid, and a BAC can be used), andcolony hybridization or plaque hybridization is conducted using a probebased on the DNA of the present invention (for example, SEQ ID NOs: 1,5, 9, 11, 13, 15, etc.). Alternatively, a genomic DNA can also beprepared by conducting PCR with primers specific to the DNA of thepresent invention (for example, SEQ ID NOs: 1, 5, 9, 11, 13, 15, etc.).A cDNA can be prepared by synthesizing a cDNA based on a mRNA extractedfrom a plant, inserting the cDNA into a vector, such as λ phage toprepare and develop a cDNA library, and conducting colony hybridizationor plaque hybridization in the same manner as above, or performing PCR.A DNA of the present invention includes not only DNA sequences of SEQ IDNOs: 1, 5, 9, 11, 13, and 15, but a DNA comprising nucleotide sequencesbased on an optional degeneracy of codons encoding amino acids of eachprotein.

A DNA of the present invention also includes, for example, a DNAencoding a protein that comprises an amino acid sequence in which one ormore amino acids are replaced, deleted, added, and/or inserted in SEQ IDNOs: 2, 6, 10, 12, 14, or 16, and has a neoxanthin cleavage activity.Thus, the DNA of the invention includes a mutant, a derivative, anallele, a variant, and a homolog of SEQ ID NOs: 1, 5, 9, 11, 13, or 15,a gene derived from a natural plant.

A modified protein encoded by the DNA of the invention comprises anamino acid sequence at least 70% (e.g., 80%, 90%, 95%, or 99%) identicalto SEQ ID NOs: 2, 6, 10, 12, 14, or 16. As used herein, “percentidentity” of two amino acid sequences or of two nucleic acids isdetermined using the algorithm of Karlin and Altschul (Proc. Natl. Acad.Sci. USA 87: 2264–2268, 1990) modified as in Karlin and Altschul (Proc.Natl. Acad. Sci. USA 90:5873–5877, 1993). Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al. (J.Mol. Biol. 215:403–410, 1990). BLAST nucleotide searches are performedwith the NBLAST program, score=100, wordlength=12. BLAST proteinsearches are performed with the XBLAST program, score=50, wordlength=3.Where gaps exist between two sequences, Gapped BLAST is utilized asdescribed in Altsuchl et al. (Nucleic Acids Res. 25: 3389–3402, 1997).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g, XBLAST and NBLAST) are used. Seehttp://www.ncbi.nlm.nih.gov.

The modified protein in which one or more amino acids are replaced inSEQ ID NOs: 2, 6, 10, 12, 14, or 16 is preferably obtained by at leastone conservative amino acid substitution. A “conservative amino acidsubstitution” is a replacement of one amino acid residue belonging toone of the following groups having a chemically similar side chain withanother amino acid in the same group. Groups of amino acid residueshaving similar side chains have been defined in the art. These groupsinclude amino acids with basic side chains (e.g., lysine, arginie,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, aspargine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

An example of a method for preparing such a DNA encoding a proteinhaving a modifiend amino acid sequence, well-known to one skilled in theart, is in vitro mutagenesis utilizing PCR (Izawa, T., “in vitromutagenesis by PCR” in Shimamoto, K. and Sasaki, T. (supervisors), CellTechnology, Supplement, Plant Cell technology Series VII, Protocols forPCR Experiments in Plants, New Edition, pp. 151–158, Shujunsha, Japan).Modification of amino acids in a protein, is ordinarily within 200 aminoacids, preferably within 100 amino acids, more preferably within 50amino acids, and further more preferably within 10 amino acids in thecase of artificial modification. Modification of an amino acid sequenceof a protein due to modification of the encoding nucleotide sequence canoccur in nature. A DNA encoding a protein having an amino acids sequencein which one or more amino acids are replaced, deleted, added, and/orinserted in an amino acid sequence encoding a wild-type neoxanthincleavage enzyme is even included in the DNA of the present invention aslong as it encodes a protein having a neoxanthin cleavage activity. TheDNA of the present invention also includes a degenerate variant in whicha mutation in a nucleotide sequence does not result in a mutation ofamino acids in a protein.

Whether a given DNA encodes a neoxanthin cleavage enzyme or not can bedetermined by expressing the DNA in E. Coli to prepare a recombinantprotein and detecting the cleavage using a cis-neoxanthin as asubstrate, according to Example 5 below.

Based on a DNA encoding a known neoxanthin cleavage enzyme, a novelneoxanthin cleavage enzyme gene can be isolated. Examples of methodswell-known by one skilled in the art for this purpose are methods usinghybridization technique (Maniatis, T et al., 1982, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y., Cold Spring HarborLaboratory Press) and polymerase chain reaction (PCR) techniques(Nakayama, H., Cell Technology, Supplement, Biological ExperimentIllustrated, Vol. 3, New Edition, Shujunsha, 1998). Specifically, oneskilled in the art can routinely isolate a DNA encoding a neoxanthincleavage enzyme gene from any plant by using a nucleotide sequence of aknown neoxanthin cleavage enzyme gene (for example, SEQ ID NOs: 1, 5, 9,11, 13, 15, etc.) or its partial sequence as a probe, as well as anoligonucleotide specifically hybridized with these sequences as aprimer. A DNA encoding a neoxanthin cleavage enzyme, capable of beingisolated by such hybridization technique or PCR technique is alsoincluded in a DNA used for improving stress tolerance in the presentinvention.

Hybridization can be performed under stringent condition by following,for example, the method described in reference (Sambrook, J., et al.,“Molecular Cloning: A Laboratory Manual” 2nd ed, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) using a [³²P ]-labeledDNA prepared by using a random prime method as a probe. A DNA is blottedto a nylon membrane and hybridized with a [³²P]-labeled fragment, forexample, in a hybrudization solution containing 30%, preferably 50%formamide, 6×SSC, 5× Denhardt's solution, and 100 μg/ml denatured salmonsperm DNA, at 37° C., preferably at 42° C. Under a stringent condition,washing is, for example, in 1×SSC, 1% SDS (room temperature), for 15 mintwice, preferably (more stringent) in 0.5×SSC, 0.5% SDS (37° C.), for 15min twice, and more preferably (further stringent) 0.1×SSC, 0.1% SDS(60° C.) for 15 min twice, and subjected to autoradiography. By“hybridizes under stringent conditions” is meant specific andnon-covalent equilibrium binding by base-pairing to an immobilizedreference nucleic acid under the above conditions.

In order to prepare a transgenic plant with improved stress toleranceusing these DNAs, the DNA is inserted into an appropriate vector, andintroduced the vector into a plant cell, and a transgenic plant isregenerated from the transformant plant cell.

As a vector used for transformation of a plant cell, any vector capableof expressing an inserted gene in the cell can be used. For example, avector comprising a promoter for constantly expressing a gene in a plantcell (for example, 35S promoter of cauliflower mosaic virus) and avector with a promoter inducibly activated by an exogenous stimulus canbe used. Alternatively, by using a promoter specific to a plant tissueto induce the expression of an objective gene, stress tolerance can beprovided specifically to a tissue highly sensitive to the stress. Forexample, an objective gene can be expressed specifically to a tissue byusing a promoter of a gene specifically expressing in seeds, such asthat of β-phaseolin gene of kidney beans (Bustos et al., EMBO J., 10:1469–1479, 1991) and that of glycinin gene of soybean (Lelievre et al.,Plant Physiol., 98: 387–391, 1992), a promoter of a gene expressingspecifically in leaves, such as that of RbcS gene of pea (Lam and Chua,Science, 248: 471–474, 1990) and that of Cab1 gene of wheat (Gotorn etal., Plant J., 3: 509–518, 1993), and a promoter of a gene expressingspecifically to roots, such as that of TobRB7 gene of tobacco (Yamamotoet al., Plant Cell, 3: 371–382, 1991), etc. Alternatively, a vectorcomprising a promoter inducibly activated by an exogenous stimulus canbe used. An example of a promoter responding to environmental stresses,such as drought, salt, or low temperature, is a promoter of rd29A gene(Yamaguchi-Shinozaki, K. and Shinozaki, K., Plant Cell, 6: 251–264,1994). A promoter to be activated by environmental stresses such asdrought and high salt concentration is also preferably used in thepresent invention. Examples of such promoters are those of ArabidopsisAtNCED3 gene, cowpea CPRD65 gene, and so on. Moreover, by using anexpression system inducible by a drug, an objective gene can beexpressed at an optional timing and in an optional tissue. An example ofan expression system induced by steroid hormone (glucocorticoid) is aninduction system using GVG gene (GAL4, VP16, Glucocorticoid receptor)(Aoyama T. and Chua, N. H., Plant J, 11: 605-12, 1997).

A vector can be inserted into any plant cells, for example, Arabidopsis(Arabidopsis thaliana), rice (Oriza sativa), tobacco (Nicotianatabacum), tomato (Lycopersicon esculentum), potato (Solanum tuberosum),maize (Zea mays), bird's foot trefoil (Lotus japonicus), and so on.Other crops or trees are also useful. A plant can be a conifer, abroad-leaved tree, a dicot, a monocot, etc. “Plant cell(s)” used hereininclude various forms of plant cells, for example, a suspended culturedcell, a protoplast, a leaf slice, a callus, etc.

For introduction of a vector into a plant cell, various methods known toone skilled in the art, for example, polyethylene glycol method,electroporation method, agrobacterium method, vacuum infiltrationmethod, particle gun method, and such can be applied. A plant can beregenerated from a transformant plant cell by methods well-known to oneskilled in the art, depending on a type of a plant cell. For example, atransformant of rice, Arabidopsis, or such can be prepared according tothe method described in “Simamoto, K., Okada K. (supervisors), CellTechnology, Supplement, Plant cell Technology Series 4, ExperimentalProtocol for Model Plants, Shujunsha, Japan.”

Once a transformant plant into which genome the DNA of the presentinvention is introduced is obtained, offspring can be obtained by sexualor asexual propagation from the plant. Alternatively, a propagationmaterial (for example, a seed, a fruit, a scion, a tuber, a tuberousroot, a stock, a callus, a protoplast, etc.) is obtained from the plant,its offspring, or clones, and the plant can be mass-produced from them.The present invention includes a plant cell into which the DNA of thepresent invention is introduced, a plant containing the cell, offspringor a clone of the plant, as well as propagation materials for the plant,its offspring, and clone.

A transformant plant produced in such a manner has an increased ABAcontent, compared with its wild type plant. Alternatively, atransformant plant has improved stress tolerance, compared with its wildtype plant. Stress tolerance can be compared by a known method. Forexample, as described in Examples below, a plant is grown under thestress condition, such as drought, high salt, low temperature, or heatcondition, and the growth of individuals is compared. For example,comparison can be made by measuring an appearance, a size of a plant orof a tissue such as a leaf, a stem, and a root, a weight (wet weight ordry weight), color, a relative growth rate, a photosynthetic activity,etc. as an index. Stress tolerance may increase in at least one tissueof a plant. A level of ABA in a plant can be determined by, for example,immunoassay, thin layer chromatography (TLC), gas chromatography (GC),and HPLC (refer to Takahashi, N. and Masuda, Y. (eds), “Plant HormoneHandbook (The Last),” Baifukan, Japan, pp. 1–21; and the referencescited therein). For example, as described in Example 7, reliablequantification is possible by finally quantifying crude purifiedfraction by HPLC with GC/MS, using labeled ABA as an internal standard.

By the present invention, a useful crop can be grown in an area exposedto environmental stress, for example, a drought zone, a cold zone, orhigh concentration of salt. In addition, the present invention can beapplied to plants other than crops for tree-planting environment.

The present invention also relates to a DNA which can decrease theexpression of a gene encoding a protein having a neoxanthin cleavageactivity to be used for lowering stress tolerance. The DNA can also beused as a reagent for decreasing stress tolerance (stress tolerancedecreasing agent). The present invention also provides uses of a DNAwhich can decrease the expression of a gene encoding a protein having aneoxanthin cleavage activity for decreasing stress tolerance.

A plant with reduced stress tolerance is useful for removing weeds andsuch from the environment, by applying to weeds and such. For example, aplant capable of inducing the decrease in stress tolerance can beprepared to apply for land improvement and such. To a plant with highregeneration ability, such as weed, a construct which inhibits theexpression of a neoxanthin cleavage enzyme gene (for example, in theantisense direction) is introduced to downstream of a promoter induciblewith a chemical (for example, glucocorticoid and so on). Thistransformant plant can normally grow without the application of thechemical. An arid land can be improved by growing the transformant weedfor several years, spraying glucocorticoid to remove the weed at once byspecifically lowering stress tolerance in the weed, and planting a cropplant. As a crop plant, a transformant crop which overexpresses aneoxanthin cleavage enzyme (a plant into which the DNA is introduced insense direction) and such can be planted.

The present inventors first successfully created a transformant plant inwhich expression of a neoxanthin cleavage enzyme is artificiallyinhibited by using a gene construct which expresses an antisense RNA ofa neoxanthin cleavage enzyme gene. This plant easily wilts in thenon-irrigated condition compared with its wild type to show the reducedstress tolerance (FIGS. 15 and 16). In such a manner, the presentinventors established a method for artificially inhibiting expression ofa gene encoding a protein having a neoxanthin cleavage activity andsuccessfully reduced stress tolerance in a plant thereby.

In the present invention, in order to reduce stress tolerance in aplant, the expression of a gene encoding a protein having a neoxanthincleavage activity can be decreased. The term “expression” of gene usedherein includes the transcription of the gene and the translation of thetranscript. The inhibition of the expression includes the completetermination of the expression. To inhibit the transcription and thetranslation of the gene encoding a protein having a neoxanthin cleavageactivity, the expression of the gene may be inhibited by targeting theDNA encoding the gene, its transcriptional control region, or thetranscript of the gene.

Any plants can be used for the present invention, and various plants canbe used. For example, Arabidopsis and such can be used. Examples of agene encoding a protein having a neoxanthin cleavage activity, which canbe a target for inhibiting the expression, are VP14 for maize (Zea mays)(Schwartz, S. H. et al., Science, 276: 1872–1874, 1997; Tan, B. V. etal., Proc. Natl. Acad. Sci. USA, 94: 12235–12240, 1997) (cDNA: SEQ IDNO: 13, protein: SEQ ID NO: 14), LeNCED1 for tomato (Lycopersiconesculentum) (Burbidge, A. et al., J. Exp. Bot., 47: 2111–2112, 1997;Burbidge, A. et al., Plant J., 17: 427–431, 1999) (cDNA: SEQ ID NO: 15,protein: SEQ ID NO: 16), AtNCED1 (cDNA: SEQ ID NO: 1, protein: SEQ IDNO: 2), AtNCED3 (cDNA: SEQ ID NO: 5, protein: SEQ ID NO: 6), and/orAtNCED5 (cDNA: SEQ ID NO: 9, protein: SEQ ID NO: 10) for Arabidopsis,CPRD65 (cDNA: SEQ ID NO: 11, protein: SEQ ID NO: 12) for cowpea, etc.Homologous genes derived from other plants can also be a target.Homologous genes of other plants can be identified and/or isolated by,for example, the hybridization method described above and such. Forreducing stress tolerance in a given plant in the present invention,using a gene or gene sequence information of another plant species (forexample, the gene above), the expression of a target gene in the desiredplant can be inhibited by known methods, such as gene silencing andantisense methods. Therefore, a target gene in an objective plant is notnecessarily isolated nor identified.

The expression of a gene encoding a protein having a neoxanthin cleavageactivity of the present invention can be inhibited by inserting a DNAfor inhibiting the expression of the gene into an appropriate vector,introducing the vector into a plant cell, and regenerating a transgenicplant from the resultant transformant cell. Any promoters can be used,for example, the promoters as described above case for improving stresstolerance. For example, use of an expression inducible type promoter canreduce stress tolerance only under a specific condition.

As a method for inhibiting the expression of a specific endogenous genein a plant, a method using the antisense technique is used the mostoften by one skilled in the art.

The antisense method is an artificial gene expression inhibition methodin which a double strand of a target mRNA with a DNA molecule (anantisense nucleic acid) complementary to the RNA transcribed from agiven gene forms for inhibiting the expression. The gene expressioninhibition method by an antisense was developed from 1960 to 1970, andin 1978, Zamecnik et al. successfully inhibited the replication andreverse transcriptase activity of chicken Rous sarcoma virus using anantisense oligomer (Zamecnik, P. C. and Stephenson, M. L., Proc. Natl.Acad. Sci. USA, 75: 280–284, 1978).

Among methods for introducing an antisense DNA, an antisense oligomer isdirectly administered into a cell, or transformation is conducted byligating an antisense DNA of a target gene with an expression vector.Examples given below demonstrate the latter method. Specifically, a cDNAof a gene encoding a protein having a neoxanthin cleavage activity isligated downstream of 35S promoter of cauliflower mosaic virus in theantisense direction to introduce the vector into a plant cell. Theantisense effect in a plant cell was first demonstrated by Ecker et al.by showing the antisense effect of the antisense RNA introduced by anelectroporation using a transient gene expression method in a plant(Ecker, J. R. and Davis, R. W., Proc. Natl. Acad. Sci. USA, 83: 5372,1986). After that, inhibition of a target gene expression by anantisense RNA expression have been reported in tobacco and petuniaplants (van der Krol, A. R. et al., Nature, 333: 866, 1988). At present,the antisense method is well established as a mean for inhibiting geneexpression in a plant. Modes of inhibition of a target gene expressionby an antisense nucleic acid include inhibition of transcriptioninitiation by the formation of a triple strand, inhibition oftranscription by the formation of a hybrid with a site at which an openloop structure is locally created by an RNA polymerase, inhibition ofsplicing by the formation of hybrid with an RNA in which a synthesis isoccurring, inhibition of splicing by the formation of a hybrid with aspliceosome formation site, inhibition of transfer from a nucleus to acytoplasm by the formation of a hybrid with an mRNA, inhibition ofsplicing by the formation of a hybrid with a capping site or a Poly (A)addition site, inhibition of translation initiation by the formation ofa hybrid with a translation initiation factor binding site, inhibitionof translation by the formation of a hybrid with a ribosome binding siteflanking an initiation codon, inhibition of extension of a peptidestrand by the formation of a hybrid with a coding region of mRNA or apolysome binding site, inhibition of gene expression by the formation ofa hybrid with an interaction site between a nucleic acid and a protein,and so on. These inhibit processes of transcription, splicing, ortranslation to inhibit the expression of a target gene (Hirajima andInoue, “New Biochemistry Experiment Lecture 2, Nucleic Acid IV,Replication and expression of a gene,” Japanese Association ofBiochemistry (eds), Tokyo-Kagakudojin, pp. 319–347, 1993).

A sequence of an antisense DNA is preferably a sequence complementary toa transcript of an endogenous gene encoding a protein having aneoxanthin cleavage activity or its part in a plant to be transformed,however, is not necessarily completely complementary as long as iteffectively inhibits the gene expression. An transcribed RNA comprisespreferably 90% or higher complementarity, and the most preferably 95% orhigher complementarity to the transcript of a target gene. Foreffectively inhibiting the expression of a target gene using anantisense sequence, a length of an antisense DNA is at least 15 or morenucleotides, preferably 100 or more nucleotides, and more preferably 500or more nucleotides. Generally, an antisense DNA to be used is shorterthan 5 kb and preferably shorter than 2.5 kb.

The expression of an endogenous gene can also be inhibited by using aDNA encoding a ribozyme. Recently, the inhibition of a gene expressionusing a DNA encoding a ribozyme has been studied. A ribozyme is an RNAcomprising an activity of catalyzing a reaction in vivo. Ribozymes havevarious activities. The studies of a ribozyme as an enzyme cleaving anRNA enable designing a ribozyme for the purpose of cleaving an RNA at aspecific site. Ribozymes include a group I intron type, huge one of 400or more nucleotides such as M1RNA included in RNaseP, and those called ahammer head type and hair pin type, comprising an active domain as longas 40 nucleotides (Koizumi, M., and Otsuka, E., Protein, Nucleic Acidand Enzyme, 35: 2191, 1990).

For example, a self-cleavage domain of a hammer head type ribozymedigests 3′ site of C15 among G13U14C15. The formation of a base pairbetween U14 and A at 9th is believed to be important for this activity,and the nucleotide at 15th can be digested if it is A or U as well as C(Koizumi, M. et al., FEBS Lett., 228: 225, 1988). If a substrate bindingsite of a ribozyme is designed to be complementary to an RNA sequenceflanking a target site, a restriction enzyme-like ribozyme whichrecognizes sequences of UC, UU, or UA in a target RNA can be created(Koizumi, M. et al., FEBS Lett., 239: 285, 1988; Koizumi, M., Otuska,E., Protein, Nucleic Acid and Enzyme, 35: 2191, 1990; Koizumi, M. etal., Nucleic Acids Res., 17: 7059, 1989). For example, hundreds of suchsites exist in a coding region of AtNCED3 gene of Arabidopsis. A hairpintype ribozyme is found in, for example, a minus strand of a satelliteRNA in tobacco ring spot virus (Buzayan, J. M., Nature, 323: 349, 1986).This ribozyme has also been shown to be able to be designed tospecifically cleave a target RNA (Kikuchi, Y. and Sasaki, N., NucleicAcids Res., 19: 6751, 1992; Kikuchi, H., (Chemistry and Biology, 30:112, 1992).

A ribozyme designed for cleaving a target is ligated with a promoter,for example, 35S promoter of cauliflower mosaic viruses and atranscription termination sequence, to be transcribed in a plant cell.When an extra sequence is added at 5′ end or 3′ end of an transcribedRNA, an activity of a ribozyme may be deleted. In this case, foraccurately excising a ribozyme portion alone from a transcribed RNAcontaining a ribozyme, another trimming ribozyme which works cis fortrimming at 5′ end and 3′ end of a ribozyme portion, can be placed(Taira, K. et al., Protein Eng., 3: 733, 1990; Dzianott, A. M. andBujarski, J. J., Proc. Natl. Acad. Sci. USA., 86: 4823, 1989; Grosshans,C. A. and Cech, R. T., Nucleic Acids Res., 19: 3875, 1991; Taira, K. etal., Nucleic Acids Res., 19: 5125, 1991). In addition, such aconstitutive unit is tandemly arranged to cleave multiple sites within atarget gene, improving the effect (Yuyama, N. et al., Biochem. Biophys.Res. Commun., 186: 1271, 1992). By using such a ribozyme, a transcriptof a target gene of this invention can be cleaved to inhibit itsexpression. Preferably, a ribozyme specifically cleaves a transcript ofa target gene. By using such a ribozyme, a transcript of a target geneof this invention is specifically cleaved to inhibit its expression.

Inhibition of the expression of an endogenous gene can also be achievedby cosuppression due to the introduction of a DNA comprising anidentical or a similar sequence to a target gene sequence.“Cosuppression” means a phenomenon in which an introduction of a genecomprising an identical or similar sequence to a target endogenous geneinto a plant by transformation inhibits the expression of both anexogenous gene introduced and a target endogenous gene. Details of themechanism of cosuppression are not clear, however, it is often observedin plants (Curr. Biol., 7: R793, 1997; Curr. Biol., 6: 810, 1996). Forexample, a plant in which the expression of a gene encoding a proteinhaving a neoxanthin cleavage activity has been cosuppressed, can beobtained by preparing a vector DNA comprising a gene encoding a proteinhaving a neoxanthin cleavage activity or a similar sequence,transforming an objective plant with the vector, and selecting amongobtained plants with a trait in which the expression of a gene encodinga protein having a neoxanthin cleavage activity has been reduced.

A gene to be used for cosuppression does not need to be completelyidentical to a target gene, however, generally has at least 70% orhigher, preferably 80% or higher, more preferably 90% or higheridentity. Genetyx (Software Development), a genetic informationprocessing software, can be used for determining an identity orcomplementarity. This program adopts Lipman-Pearson method (Lipman, D.J. and Pearson, W. R., Science, 227: 1435–1441, 1985). This method firstcompares sequence data, and calculates identity among the sequences withhigh homology in consideration with a deletion of a sequence (GAP).

A transformant plant with reduced stress tolerance can be prepared byusing a DNA as described above to be used in the present invention thatinhibits the expression of a gene. Specifically, the DNA is insertedinto an appropriate vector, the vector is introduced into a plant cell,and a transgenic plant is regenerated from the transformant plant cell.As a vector to be used, any vectors can be used in the same manner as inthe above case of increasing the stress tolerance as long as an insertedgene can be expressed in a plant cell. Any plant cells can be used toinsert a vector. A plant can be a conifer, a broad-leaved tree, a dicot,a monocot, etc. “Plant cell(s)” referred herein include various forms ofplant cells, for example, a suspended cultured cell, a protoplast, aleaf slice, a callus, and so on.

Introduction of a vector into a plant cell, and regeneration of a plantfrom a transformant cell, can be performed by a method known to oneskilled in the art, depending on a type of plant cells in the samemanner as in the case of improving stress tolerance. Once a transformantplant in which a DNA of the present invention is introduced into thegenome can be obtained, offspring can be obtained from the plant bysexual or asexual propagation. Alternatively, a propagation material(for example, a seed, a fruit, a scicon, a tuber, a tuberous root, astock, a callus, a protoplast, etc.) can be obtained from the plant, itsoffspring, or clones, and the plant can be mass-produced from them. Thepresent invention includes a plant cell in which a DNA of presentinvention is introduced, a plant containing the cell, offspring or aclone of the plant, as well as a propagation material for the plant, itsoffspring, and clone.

The transformant plant created in such a manner has a reduced ABAcontent, compared with the wild type plant. Alternatively, thetransformant plant has a decreased stress tolerance compared with thewild type plant. The present invention can be applied to, for example, aweed to effectively eliminate it. In addition, using a induciblepromoter as described above, a transformant plant of the presentinvention can be used for land improvement and such.

The present invention enables creating a plant in which stress tolerancehas been increased or decreased. A plant with the improved stresstolerance can grow in harsh land where plants cannot grow thus far.Reduction of stress tolerance can be applied to weeds and such to beeliminated. The method of the present invention can be applied toagriculture to expand cultured area and increase crop yields.

Any patents, patent applications, and publications cited herein areincorporated by reference.

The present invention is illustrated in detail below with reference toexamples, but is not to be construed as being limited thereto.Experimental conditions used in the present Examples are as follows.

Growth of Cowpea

Seeds of Cowpea (Vigna unguiculata IT84S-2246-4) were sown in pots andgrown for 8 days in a greenhouse with a photoperiod of 16 hours (inaddition to natural light, artificial lighting was supplemented whenillumination was insufficient), temperature of 25° C., and appropriatewatering.

Dehydration Treatment

For dehydration treatment, plants were pulled out of the pot carefullyto avoid injury, weighed, and dehydrated on Whatman 3 MM filter paper atroom temperature and approximately 60% humidity under dim light (300lux). For the control, plants were pulled out of the pot and immediatelytransplanted in well-watered soil that was maintained under the samecondition for dehydration treatment group.

Analysis of DNA Sequence

Plasmid DNA templates were prepared using the Automatic PlasmidIsolation System Model PI-100 (KURABO) and sequenced using the DNASequencer Model 373A (ABI). Nucleotide sequences and amino acidsequences were analyzed using a GeneWorks Software System(IntelliGenetics, Inc.), Sequencher 3.0 (Hitachi Software), and theUniversity of Wisconsin Genetic Computer Group (GCG) program.

EXAMPLE 1 Isolation of cDNA Clones Corresponding to Genes Induced byDehydration

A cDNA library was constructed with poly(A)⁺ RNA that had been isolatedfrom 8-day-old cowpea plants after dehydration stress for 10 hours asfollows.

Whole plants were harvested, washed gently to remove soil from the rootsand dehydrated on Whatman 3 MM filter paper at room temperature andapproximately 60% humidity under dim light for 10 hours. Total RNA wasprepared from the plants after dehydration treatment by theaforementioned method (Nagy, F. et al., “Analysis of gene expression intransgenic plants. ” In Gelvin and Schilperoort (eds), “Plant MolecularBiology Manual. B4.,” Kluwer Academic Publishers, Dordrecht, pp. 1–29,1988). By following the reference (Sambrook, J. et al., “MolecularCloning: A Laboratory Manual,” 2nd ed, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989), total RNA was passed through anOligo-dT cellulose column twice to prepare poly(A)⁺ RNA. About 2% of theRNA applied to the column was collected as the Poly (A)⁺ RNA fraction.Double-stranded cDNA was synthesized from the Poly (A)⁺ RNA using cDNASynthesis System Plus (Amersham Pharmacia Biotech). A cDNA library wasconstructed from the cDNA using cDNA Cloning System (Amersham PharmaciaBiotech).

The cDNA library was differentially screened with cDNA prepared frompoly(A)⁺ RNA that had been isolated from unstressed cowpea plants andwith cDNA prepared from poly(A)⁺ RNA that had been isolated from plantsafter dehydration stress for 10 hours. By following the reference(Sambrook, J. et al., “Molecular Cloning: A Laboratory Manual,” 2nd ed,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),plaque hybridization was conducted to screen 1×10⁴ plaques from the cDNAlibrary.

As a result, plaques giving a stronger hybridization signal with[³²P]-labeled cDNA were obtained from 10-hour dehydrated cowpea plants.The plasmid regions of the phage clones were excised in vivo and used totransform Escherichia coli cells. The cDNA fragments from the resultantplasmids were analyzed using the restriction map and the bordersequences of the cDNA fragments. From these analyses, cDNA was grouped,and a cDNA clone, named CPRD (Cow Pea Responsive to Dehydration) 65could be identified.

Dehydration-induced expression of the gene corresponding to the CPRD65clone was analyzed by Northern blot hybridization. The 8-day-old plantswere pulled out of the soil and dehydrated for various periods up to 12hours. As controls, similar cowpea plants were pulled out of the soiland were immediately transplanted to well-watered soil. Total RNA wasthen isolated from dehydrated or control plants for Northern blothybridization.

Total RNA was isolated according to the method of Nagy et al. (Nagy, F.et al., “Analysis of gene expression in transgenic plants.” In Gelvinand Schilperoort (eds), “Plant Molecular Biology Manual, B4.,”KluwerAcademic Publishers, Dordrecht, pp, 1–29, 1988), fractionated in a 1%agarose gel containing formaldehyde, and transferred to a nylon filter(Sambrook, J. et al., “Molecular Cloning: A Laboratory Manual,” 2nd ed,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).The nylon filter was hybridized with [³²P]-labeled CPRD65 cDNA fragmentsin 50% formamide, 5×SSC, 25 mM sodium phosphate buffer (pH 6.5), 10×Denhardt's solution, and 250 μg/ml of denatured salmon sperm DNA at 42°C. The filter was washed twice with 0.1×SSC, 0.1% SDS at 60° C. for 15min, and subjected to autoradiography.

FIG. 1 shows the time course of induction of the expression thatcorresponds to the CPRD65 gene against dehydration treatment. Theexpression of CPRD65 was significantly increased by dehydration stress.The mRNA corresponding to the CPRD65 was observed to accumulate within 2hours after the initiation of dehydration treatment.

Cowpea plants dehydrated for 10 hours appeared wilted. These wiltedplants showed recovery from wilting within 4 hours after transfer towell-watered soil (rehydration treatment). After rehydration, the levelof CPRD65 mRNA decreased (FIG. 1). The CPRD65 gene exhibited typical andsignificant responses to drought stress, namely, induction of thetranscriptions by dehydration and reduction of the level uponrehydration. These facts suggested that the CPRD65 gene is involved indrought tolerance.

EXAMPLE 2 Sequence Analysis of the CPRD65 cDNA

Since the CPRD65 cDNA fragment isolated in Example 1 was not possiblyfull length, the same cDNA library was screened again with a partialCPRD65 cDNA as a probe to isolate a full-length cDNA (SEQ ID NO: 11). Anamino acid sequence encoded by the full-length cDNA clone (SEQ ID NO:12) was shown in FIG. 2. The full-length CPRD65 cDNA consists of 2432bp, including a 5′-flanking region of 125 bp and 3′-flanking region of486 bp. One polyadenylation consensus sequence (AATAAA) was found in the3′-flanking region. This sequence has an open reading frame encoding apolypeptide of 612 amino acids with a calculated molecular weight forthe putative protein of 67.6 kDa. Comparison of the deduced amino acidsequence of the CPRD65 protein with the protein database revealed anextensive homology with VP14 from maize (Zea mays) (61%) (Schwartz, S.H., Science, 276: 1872–1874, 1997) and a neoxanthin cleavage enzyme fromtomato (Lycopersicon esculentum) (69%), (Burbidge, A. et al., J. Exp.Bot., 47: 2111–2112, 1997; Burbidge, A. et al., Plant J., 17: 427–431,1999) as shown in FIG. 2. The putative CPRD65 protein seems to contain atransit polypeptide in its N-terminal region like the VP14 protein. TheN-terminal sequences of the CPRD65, VP14, and tomato neoxanthin cleavageenzyme have low sequence similarity, but structural similarity.

EXAMPLE 3 Genomic Southern Blot Analysis of the CPRD65 Gene

In order to analyze genes related to the CPRD65 of cowpea plants,genomic Southern blot hybridization was conduced in the conditions oftwo stringencies (FIG. 3).

The genomic Southern blot analysis was conducted according to the methodof the reference (Sambrook, J. et al., “Molecular Cloning: A LaboratoryManual,” 2nd ed, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989). Genomic DNA of 10 μg was digested with restrictionenzymes, separated in a 1% agarose gel, and blotted to a nylon filter.The filter was hybridized with [³²P]-labeled fragments in 30% formamide,6×SSC, 5× Denhardt's solution, and 100 μg/ml of denatured salmon spermDNA at 42° C. The filter was washed twice with 0.1×SSC, 0.1% SDS at 60°C. for 15 min (B), or washed twice with 0.5×SSC, 0.5% SDS at 37° C. for15 min (A), and subjected to autoradiography.

The CPRD65 cDNA had no internal restriction site for EcoRI and XbaI andhad two flanking internal restriction sites for HindIII, confirmed byits nucleotide sequence. One hybridized band in the EcoRI and XbaIdigest and two hybridized bands in the Hind III digest were detectedusing the CPRD65 cDNA as a probe. Some additional faint hybridized bandswere detected under the above stringency condition (A). These resultssuggest that the CPRD65 gene constitutes a small gene family withrelated genes.

EXAMPLE 4 Northern Blot Analysis of the CPRD65 Gene

The effects of various environmental stresses on the expression of theCPRD65 gene were analyzed. For high salinity, ABA, and water treatments,plants were pulled out of the soil in the same manner as in thedehydration treatment, and grown by the hydroculture in the solutionscontaining 250 mM NaCl, 100 μM ABA, and deionized water, respectively.For heat and cold treatments, potted plants were transferred to theincubators at 40° C. and 4° C., respectively. Each stress treatment toplants was conducted for 0, 1, 2, 5, 10, and 24 hours. After thetreatments, the treated plants were immediately frozen with liquidnitrogen, and the RNAs were isolated for Northern blot analysis.

As a result, it was found that the expression of this gene was stronglyinduced under a high-salt condition, but not by cold or heat stress(FIG. 4A). The induction of the CPRD65 gene was not detected by ABAtreatment or water treatment.

To determine the tissue specificity of the expression of the CPRD65 geneunder drought stress, Northern blot hybridization of total RNA preparedfrom leaves, stems, or roots under a normal or drought condition wasperformed (FIG. 4B). The CPRD65 transcript was strongly induced in stemsand leaves by drought treatment, but less in roots.

EXAMPLE 5 Enzymatic Activity of the Bacterially Expressed CPRD65 Protein

The deduced amino acid sequence of the CPRD65 gene has high homologywith an amino acid sequence of a neoxanthin cleavage enzyme encoded bythe maize VP14 gene (FIG. 2). To examine whether the CPRD65 gene encodesa neoxanthin cleavage enzyme, the biochemical properties of therecombinant CPRD65 protein expressed in E. coli were analyzed. A DNAfragment for the CPRD65 coding region was amplified by PCR and fused tothe GST gene in frame using the pGEX4T-1 (Pharmacia) to construct achimeric plasmid pGST-CPRD65 as follows.

The DNA encoding the CPRD65 protein was amplified by PCR using primers:5′-ATTGAATTCATGCCTTCAGCTTCAAAC-3′ (SEQ ID NO: 19) and5′-ATTGGATCCCAAAAGCTACACGCTGGTCCCC-3′ (SEQ ID NO: 20). The PCR fragmentwas inserted into the EcoRV site of pBluescript II SK⁺ vector. Sequencesof the inserted PCR fragments were confirmed to determine whether amutation was generated in a nucleotide sequence by PCR. The PCR fragmentin which any mutation was not identified in the nucleotide sequence wasisolated from the pbluescript II SK⁺ vector as a DNA fragment usingrestriction enzymes (EcoRI and XhoI) and inserted into the EcoRI to XhoIsite of pGEX4T-1 (Amersham Pharmacia Biotech) to construct pGST-CPRD65.Cells of Escherichia coli strain JM109 were transformed with pGST-CPRD65or pGEX4T-1 and cultured in L broth at 37° C. When OD₆₀₀ reached about0.5, isopropyl β-D-thiogalactopyranoside (IPTG) was added, andincubation was continued for 12 hours at 17° C. The cells wereharvested, washed, and suspended in extraction buffer [10 mM Tris-HCl(pH 8.0), 5 mM MgCl₂, 5% glycerol, 0.1 mM phenylmethylsulfonyl fluoride(PMSF), and 0.1 mM dithiothreitol (DTT)]. The procedures forpurification of the fused protein and digestion with thrombin wereperformed according to the instruction manual for the GST gene fusionsystem (Amersham Pharmacia Biotech). The protein concentration wasdetermined with a protein assay kit (Bio-Rad).

The GST-CPRD65 fusion protein was overexpressed in E. coli in the mannerdescribed above, and purified from the crude cell extract using aglutathione-Sepharose 4B. Whether this purified GST-CPRD65 recombinantprotein digests cis-neoxanthin, trans-violaxanthin, and cis-violaxanthinto produce xanthoxin was examined.

The assay procedures for neoxanthin cleavage enzyme activity have beendescribed (Schwartz, S. H. et al., Science, 276: 1872–1874, 1997).cis-Neoxanthin and trans-violaxanthin were prepared from spinach leaves.cis-Violaxanthin was prepared from orange peel. The reaction mixture(100 μl) contained 100 mM Bis-Tris (pH 6.7), 0.05% Triton X-100, 10 mMascorbic acid, 5 mM FeSO₄, and a protein sample. The reaction wasallowed to proceed at room temperature for 1 hour. After addition of 1ml of water, the reaction mixtures were extracted with n-hexane (1 ml×2)and then ethyl acetate (1 ml×2). The n-hexane fraction was concentratedand submitted to HPLC analysis on a column of Nucleosil 5 C₁₈ (150 mmlength, 8 mm internal diameter (i. d.)). The column was eluted with alinear gradient between solvent A (85% ethanol) and solvent B(chloroform and methanol, 1:1) at a flow rate of 1.5 ml/min. Theconcentration of solvent B was increased from 10% to 50% in 25 min, andkept at 50% for 5 min. The absorbance of the eluate was monitored with aUV detector at 440 nm. The ethyl acetate fraction was purified with HPLCon a column of Nucleosil 5 C₁₈ (150 mm length, 8 mm i. d.). The columnwas eluted with 50% aqueous methanol at a flow rate of 1.5 ml/min, andthe absorbance of the eluate was monitored with a UV detector at 260 nm.The predicted xanthoxin fraction was collected and submitted to GC-MSanalysis. In each step, samples were shielded from light as much aspossible.

GC-MS analysis was conducted as follows. An AUTOMASS mass-spectrometer(Nippon Denshi) equipped with a 5890 gas chromatography (HewlettPackard) was used for the analysis. The analytical conditions were asfollows: ionization, EI 70 eV; column, DB-5 (15 m length; 0.25 mm i. d.;0.25 μm film thickness; J&W Scientific); carrier gas, He (1 ml min⁻¹);injection temperature, 250° C.; transfer line temperature, 250° C.; andinitial heating temperature, 80° C. Starting 1 min after injection, theheating temperature was increased to 200° C. at a rate of 30° C.min⁻¹followed by further increment to 230° C. at a rate of 5° C. min⁻¹.

As shown in FIG. 5, the predicted C25-product and xanthoxin weredetected in the reaction mixture with the GST-CPRD65 protein andcis-neoxanthin by HPLC analysis. The occurrence of xanthoxin wasconfirmed by GC-MS analysis in which ions characteristic to xanthoxinwere observed. The ions and their relative intensities were: m/z 250(4), 168 (32), 149 (77), 107 (61), and 95 (100). Xanthoxin andC25-product were not formed from trans-violaxanthin (data not shown).These results were not affected by the treatment with thrombin whichseparates the GST-CPRD65 recombinant protein into GST and CPRD65portions.

EXAMPLE 6 Analysis of N Terminal Region of the CPRD65 Protein as aTransit Peptide in Protoplasts Prepared From Arabidopsis

The N-terminal region of the CPRD65 protein has typical structuralfeatures of transit peptides that are involved in chloroplast targeting.This structural feature of the CPRD65 protein suggests that the matureCPRD65 protein is located in plastids including chloroplasts. To analyzethe role of its N-terminal region as a transit peptide, a chimeric gene35S::CPRD65N-sGFP that encodes the N-terminal region of the CPRD65protein (1–148) between the CaMV 35S promoter and the synthetic greenflorescent protein (sGFP) gene of the jellyfish Aequorea victoria (Chiu,W., etal., Curr. Biol., 6:325–330,1996) was constructed.

The DNA corresponding to the N-terminal peptide (1 to 148 amino acids)of the CPRD65 protein was amplified by PCR using primers:5′-ATATATCTAGAATGCCTTCATCAGCTTCAAACACTTGG-3′ (SEQ ID NO: 21) and5′-ATATAGGATCCCTCCGGCACCGGCGCGAAGTTCCCG-3 (SEQ ID NO: 22). The PCRfragment was inserted into the pBluescript II SK⁺vector and verified tohave no sequence mutation caused by PCR. The DNA fragment was insertedinto the site between 35S-promoter and SGFP gene on transient expressionvector (Chiu, W. et al., Curr. Biol., 6: 325–330, 1996). Thepreparation, DNA transfection, and incubation of the Arabidopsisprotoplasts were performed as previously described (Abel, S. andTheologis, A., Plant J., 5: 421–427, 1994). 35S::CPRD65N-sGFP fusionconstruct and its control construct (35S::sGFP) were introduced intoprotoplasts prepared from Arabidopsis by a DNA-transfection method(Abel, S. and Theologis, A., Plant J., 5: 421–427, 1994). Theprotoplasts were observed by fluorescent microscopy 2 to 4 days afterthe transformation. As shown in FIG. 6, when 35S::CPRD65N-sGFP wastransiently expressed in the protoplasts, fluorescence was localized inplastids. On the other hand, when 35S::sGFP construct was introduced,fluorescence was detected not in plastids, but mainly in the cytoplasm.These results suggest that N-terminal region of the CPRD65 proteinfunctions as a transit peptide to target the CPRD65 protein into theplastids. The CPRD65 protein was expected to be localized in plastids,and function to produce ABA in plastids.

EXAMPLE 7 Accumulation of ABA by Dehydration Stress in 8-day-old CowpeaPlants.

The accumulation of endogenous ABA level in a 8-day-old cowpea plant wasmeasured by dehydration conditions.

Samples were homogenized in liquid nitrogen and extracted with aqueousmethanol (20 to 80%) twice. After addition of [²H₃]ABA, the extractswere concentrated, and submitted to a standard solvent fractionationprocedure to give an acidic-ethyl acetate soluble fraction. It waspurified using Bond Elut cartridge (C₁₈ and DEA, Varian) by theprocedure reported previously (Wijayanti, L., et al., Biosci. Biotech.Biochem., 59: 1533–1535, 1995). Purified samples from undesiccatedplants were hen subjected to HPLC analysis with a Senshu Pak ODS-2101-Ncolumn (100 mm length, 6 mm i.d., Senshu Scientific Co.). The analyticalconditions were the same as reported previously (Wijayanti, L., et al.,Biosci. Biotech. Biochem., 59: 1533–1535, 1995). Samples thus purifiedwere methylated with etherial diazomethane and submitted to GC-SIManalysis.

As shown in FIG. 7, ABA began to accumulate within 2 hours afterdehydration. The level of ABA in 10-hour dehydrated plants was 140 timeshigher than that in unstressed control plants. The timing ofaccumulation of the CPRD65 mRNA was earlier than that of ABA mRNAaccumulation (FIG. 7).

The expression of the CPRD65 gene was strongly induced by drought stressin leaves and stems, but slightly in roots (FIG. 4B). The relationshipbetween the expression of the CPRD65 gene and the accumulation ofendogenous ABA under drought stress was examined. The 8-day-old cow peaplants were separated into leaves, stems, and roots, and thendehydrated. The endogenous ABA levels in these organs were measuredbefore or after dehydration treatment. As shown in FIG. 8, endogenousABA were dramatically accumulated by drought stress in leaves and stems,but slightly in roots. The tissue-specific pattern of ABA accumulationunder drought stress was consistent with that of the expression of theCPRD65 gene as shown in FIGS. 4B and 8.

EXAMPLE 8 Analysis of Xanthophylls in Cowpea Leaf

Xanthophylls in cowpea leaf were analyzed to find possible substancesfor the CPRD65 protein.

Samples were extracted with acetone twice, and the extracts wereconcentrated, dissolved in 80% methanol (1 ml), and loaded onto a BondElut C₁₈ column. The column was washed with additional 4 ml of 80%methanol, and xanthophylls were eluted with 5 ml ofmethanol-water-chloroform (71:9:20). The eluate was concentrated andapplied to HPLC analyses with columns of Nucleosil 5 C₁₈ and Senshu PakSilica-2251-S (250 mm length, 6 mm i. d.). Conditions for ODS-HPLC werethe same as described above. For Silica-HPLC, a flow rate of 1.5 ml/minand a linear gradient of solvent B concentration from 10% to 100% in 30min were used where solvent A was ethyl acetate-n-hexane (1:1) andsolvent B is ethyl acetate. The xanthophylls were identified from theirvisible and ultraviolet spectroscopic data.

trans-Neoxanthin, trans-violaxanthin, and cis-neoxanthin were detectedas major xanthophylls, and cis-violaxanthin was detected as a minorcomponent in cowpea leaf by optical spectroscopic analysis of visibleand ultraviolet lights (data not shown). The endogenous amounts oftrans-neoxanthin, trans-violaxanthin, and cis-neoxanthin were notsignificantly different between under normal growth conditions anddrought conditions.

As shown above, cowpea drought-inducible CPRD65 gene encodes theneoxanthin cleavage enzyme, and its product is localized in plastids.The CPRD65 gene was strongly induced mainly in leaves and stems underdrought and high salt conditions. Strong accumulation of ABA in leavesand stems under drought conditions was observed, which showed a similarpattern of the CPRD65 gene expression. These results strongly suggestthat the CPRD65 protein is an enzyme mainly involved in the ABAbiosynthesis under drought stress in cowpea plants.

EXAMPLE 9 Isolation of Arabidopsis cDNA Clone Encoding a Homolog ofNeoxanthin Cleavage Enzyme Gene

Using the CPRD65 as a probe, a cDNA library of Arabidopsis (Abe H. etal, Plant Cell, 9: 1859–1868, 1997) was screened. As a result, manyplaques indicating strong hybridization signals were obtained. Fromthese plaques, phage clones were isolated, and a cDNA region was excisedwith restriction enzymes to insert it into a pBluescript SK+andtransfect E. coli. By analysis of a DNA sequence, these clones wereclassified into one group. This was designated AtNCED3. The AtNCED3showed a significant homology with the CPRD65 encoding a neoxanthincleavage enzyme (FIG. 9). A nucleotide sequence of the AtNCED3 cDNA andan amino acid sequence of the AtNCED3 protein are shown in SEQ ID NOs: 5and 6, respectively.

EXAMPLE 10 Isolation of the AtNCED1, 2, 4, and 5, and Analysis ofPhylogenic Tree

Search on DNA databases using the nucleotide sequences of the CPRD65 andthe AtNCED3 identified four sequences with high homology (Ac. No.AL021713, AL021687, AJ005813, AB028621).

In order to isolate a gene with high homology existing in the AL021713sequence, a target gene fragment was amplified by the PCR method using agDNA as a template, and 5′-CCCGGGATCCCTCAAGCCTCTCTATACCG-3′ (SEQ ID NO:23) and 5′-CCCGGGATCCTTTATACGGATTCTGAGGGAG-3′ (SEQ ID NO: 24) asprimers. Using the fragment as a probe, a clone containing the targetgene was isolated from the gDNA library (Clontech). The gene wasamplified again by the PCR method using5′-CCCGGGATCCCTCAAGCCTCTCTATACCG-3′ (SEQ ID NO: 23) and5′-CCCGGGATCCTTTATACGGATTCTGAGGGAG-3′ (SEQ ID NO: 24) as primers, andcloned into the EcoRV site of pBluescript II SK⁺ (Stratagene) todetermine a nucleotide sequence. This gene was designated AtNCED1. Anucleotide sequence of the AtNCED1 cDNA and an amino acid sequence ofthe AtNCED1 protein are shown in SEQ ID NOs: 1 and 2, respectively.

In order to isolate a gene with high homology existing in the AL021687sequence, a target gene fragment was amplified by the PCR method using agDNA as a template, and 5′-ATTGAATTCATGGACTCTGTTTCTTCTTCTTCC-3′ (SEQ IDNO: 25) and 5′-ATTGAATTCTTAAAGCTTATTAAGGTCACTTTCC-3′ (SEQ ID NO: 26) asprimers. Using the fragment as a probe, a clone containing the targetgene was isolated from the gDNA library (Clontech). The gene wasamplified again by the PCR method using5′-ATTGAATTCATGGACTCTGTTTCTTCTTCTTCC-3+ (SEQ ID NO: 25) and5′-ATTGAATTCTTAAAGCTTATTAAGGTCACTTTCC-3′ (SEQ ID NO: 26) as primers, andcloned into the EcoRV site of pBluescript II SK⁺ (Stratagene) todetermine a nucleotide sequence. This gene was designated AtNCED2. Anucleotide sequence of the AtNCED2 cDNA and an amino acid sequence ofthe AtNCED2 protein are shown in SEQ ID NOs: 3 and 4, respectively.

In order to isolate a gene with high homology existing in the AJ005813sequence, a target gene fragment was amplified by the PCR method using agDNA as a template, and 5′-AAGAAmTCATGGCGGAGAAACTCAGTGATGGCAGC-3′ (SEQID NO: 27) and 5′-AAAAGAATTCGGCTTATATAAGAGTTTGTTCCTGG-3′ (SEQ ID NO: 28)as primers. Using the fragment as a probe, a clone containing the targetgene was isolated from the cDNA library (Clontech). The gene wasamplified again by the PCR method using5′-AAGAATTCATGGCGGAGAAACTCAGTGATGGCAGC-3′ (SEQ ID NO: 27) and5′-AAAAGAATTCGGCTTATATAAGAGTTTGTTCCTGG-3′ (SEQ ID NO: 28) as primers,and cloned into the EcoRV site of pBluescript II SK⁺ (Stratagene) todetermine a nucleotide sequence. This gene was designated AtNCED4. Anucleotide sequence of the AtNCED4 cDNA and an amino acid sequence ofthe AtNCED4 protein are shown in SEQ ID NOs: 7 and 8, respectively.

In order to isolate a gene with high homology existing in the AB028621sequence, DNA was isolated from P1 clone MUJ8. The gene was amplified bythe PCR method using 5′-CGGGATCCATGCAACACTCTCTTCGTTCTGATCTTCTTC-3′ (SEQID NO: 29) and 5′-CGGGATCCTCAGAAAACTTGTTCCTTCAACTGATTCTCGC-3′ (SEQ IDNO: 30) as primers, and cloned into the EcoRV site of pBluescript IISK+(Stratagene) to determine a nucleotide sequence. This gene wasdesignated AtNCED5. A nucleotide sequence of the AtNCED5 cDNA and anamino acid sequence of the AtNCED5 protein are shown in SEQ ID NOs: 9and 10, respectively.

FIG. 10 shows the alignments of the amino acid sequences of the AtNCED1to 5. To examine relationship between amino acid sequences deduced fromeach sequence and sequences on the databases, the phylogenic treeanalysis was conducted (FIG. 11). A phylogenic tree was constructedusing GeneWorks (Intelligenetics, Inc.), a software for analyzing genes.Algorithm used UPGMA (Unweighted Pair Group Method with Arithmetic Mean:Molecular Evolutionary genetics, written by Nei, M., translated by Gojo,H. and Saito N., Baifukan, pp. 252–256, Japan).

EXAMPLE 11 Northern Blot Analysis of the AtNCED Genes

Effects of various environmental stresses on the expression of eachidentified AtNCED gene were analyzed by Northern blot analysis.

Plants grown on an agar plate for three weeks were used for each stresstreatment. For dehydration stress, plants were pulled out of an agarmedium, and air-dried on filter paper (relative humidity 50%). For saltstress, ABA treatment, and water treatment as a control, plants werepulled out and placed in a Petri dish containing 250 mM NaCl solution,100 μM ABA solution, and distilled water, respectively, so that onlyroots were immersed, for a certain period of time at room temperaturewith a lid closed. For cold and heat stresses, agar plates were placedin a constant-temperature incubator at 4° C. and 40° C., respectively,for a certain period of time.

The plants treated with each environmental stress above were crushed inliquid nitrogen, total RNA was extracted (Nagy F, Kay SA and Chua N-H(1988) Analysis of gene expression in transgenic plants. In Gelvin andSchilperoort (eds), Plant Molecular Biology Manual, B4. Kluwer AcademicPublishers, Dordrecht, pp 1–29), and 20 μg each of samples were loadedon each lane of a 1% agarose gel and electrophoresed. The RNA wasblotted from the gel to a nylon membrane after the electrophoresis, andsubjected to Northern hybridization using a [³²P]-labeled cDNA probe(Sambrook, J., Fritsch EF and Maniatis T (1989) Molecular Cloning: ALaboratory manual, 2nd ed, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.).

As a result, it was found the AtNCED3 gene expression was stronglyinduced by drought, high salt concentration, and cold condition. Heatcondition did not induce the expression. In addition, for ABA treatmentor water treatment, the induction of the AtNCED3 gene expression was notdetected (FIG. 12).

Example 12 Enzymatic Properties of the Bacterially Expressed AtNCEDProtein

The deduced amino acid sequence of the AtNCED3 gene has high homologywith that of the cowpea CPRD65 gene encoding a neoxanthin cleavageenzyme (FIG. 9). To examine whether the AtNCED3 gene encodes aneoxanthin cleavage enzyme, the biochemical properties of therecombinant AtNCED3 protein expressed in E. coli were analyzed.

The DNA encoding the AtNCED3 protein was amplified by PCR using thecloned AtNCED3 cDNA as a template, and5′-ATTGAATTCATGGCTTCTTTCACGGCAACGGC-3′ (SEQ ID NO: 31 and5′-GTTTTCCCAGTCACGAC-3′ (SEQ ID NO: 32) as primers. The PCR fragment wascloned into the EcoRV site of pBluescript II SK⁺ (Stratagene). Sequencesof the inserted PCR fragments were confirmed to determine whether amutation was generated in a nucleotide sequence by PCR. The DNA fragmentin which any mutation was not identified was cloned in frame into theEcoRI site of pGEX4T-1 containing glutathione S-transferase (GST) gene(Amersham Pharmacia Biotech) to construct chimeric plasmid pGST-AtNCED3.Cells of Escherichia coli strain JM109 were transformed withpGST-AtNCED3 or pGEX4T-1 and cultured in L broth at 37° C. When CD₆₀₀reached about 0.5, isopropyl β-D-thiogalactopyranoside (IPTG) was added,and incubation was continued for 12 hours at 17° C. The E. coli cellswere harvested, washed, and suspended in extraction buffer [10 mMTris-HCl (pH 8.0), 5 mM MgCl₂, 5% glycerol, 0.1 mM phenylmethylsulfonylfluoride (PMSF), and 0.1 mM dithiothreitol (DTT)]. The procedures forpurification of the fused protein and digestion with thrombin wereperformed using glutathione-Sepharose 4B [the GST gene fusion system(Amersham Pharmacia Biotech)] according to its instruction manual. Theprotein concentration was determined with a protein assay kit (Bio-Rad,CA, USA).

As a result of assays for the neoxanthin cleavage enzyme activity,expected C25 product and xanthoxin were detected in the reaction mixturecontaining GST-AtNCED3 protein and cis-neoxanthin, confirming that theAtNCED3 protein comprises a neoxanthin cleavage activity. The similarexperiment detected a neoxanthin cleavage activity in the AtNCED1 andAtNCED5.

EXAMPLE 13 Preparation of Transgenic Plants

Arabidopsis (Arabidopsis thaliana (L.) Heynh. ecotype Columbia) was usedas a sample. Wild type Arabidopsis plants were sown in a 9 cm-diameterplastic-pot with culture soil, grown for 6 weeks at 22° C. with aphotoperiod of 16-hours, and then used for transformation.

A vector without a GUS reporter gene (pBE2113NOT) was constructed from apBE2113 vector with a kanamycin resistant marker and a 35S promoter ofcauliflower mosaic virus (Mitsuhara, I. et al., Plant Cell Physiol., 37:49–59, 1996), and the cDNA of the AtNCED3 isolated from Arabidopsis wasligated to the vector at BamHI site in the right direction (a sensedirection) or the opposite (an antisense direction). The obtainedvectors were introduced into a soil bacterium (Agrobacterium tumefaciensstrain GV3101 (pMP90)) by mixing the vectors with the bacterium. TheAgrobacterium tumefaciens with the target gene was selected by kanamycin(Km) resistance, and infected wild type Arabidopsis plants using thevacuum infiltration method (Bechtold, N. et al., C. R. Acad. Sci. Paris,Life Sci., 316: 1194–1199, 1993). From the infected plants, dry seedswere harvested, sown on an agar plate supplemented with Km, and grown toselect individuals of the transformant first generation (T1). Seeds oftransformant second generation (T2) obtained from the transformant firstgeneration were sown on an agar plate supplemented with Km, and grown tocollect seeds for the third generation (T3) from plants showing Kmresistance. Moreover, seeds of the third generation were sown on a platesupplemented with Km in the similar manner, and those of all seedsshowing drug resistance were used for the following experiments as a T3homologous line. Finally, two lines for each sense and antisensetransformant of the AtNCED3 gene were isolated.

EXAMPLE 14 Evaluation of the Expression of the AtNCED3 Gene inTransformants

The expression of the AtNCED3 gene in wild type Arabidopsis and AtNCED3gene-transformant plants was evaluated by Northern hybridization method.

Plants cultivated for a month were used for analyses of the AtNCED3 geneexpression in transformants. The plants were pulled out and air-dried onfilter papers as drought stress (relative humidity 50%). Plants thatbore the environmental stress treatment above were broken in liquidnitrogen to extract total RNA (Nagy, F., Kay, S. A. and Chua, N. -H.(1988) Analysis of gene expression in transgenic plants. In Gelvin andSchilperoort, eds, Plant Molecular Biology Manual, B4. Kluwer AcademicPublishers, Dordrecht, pp 1–29). The RNA was electrophoresed (20 μg perlane) on 1% agarose gel, transferred from the electrophoresed gel onto anylon membrane, and subjected to Northern hybridization by using a[³²P]-labeled RNA probe (Sambrook, J., Fritsch, E. F. and Maniatis, T.(1989) Molecular Cloning: A Laboratory Manual, 2nd ed, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.).

As a result, the AtNCED3 gene was not expressed in the wild type beforethe drought stress but already expressed in the sense plants. On theother hand, after the drought treatment, the AtNCED3 gene was induced toexpress by drought in the wild type plants but not expressed in theantisense plants even after the drought stress treatment (FIG. 13).

EXAMPLE 15 Evaluation of Endogenous ABA Amount in Transformants

Endogenous ABA amounts were measured in wild type Arabidopsis andAtNCED3 gene-transformant plants.

Plants cultivated for a month were used for evaluation of endogenous ABAamounts in wild type Arabidopsis and transformants. Samples werehomogenized in liquid nitrogen and extraction with aqueous methanol (20to 80%) was performed twice. After adding [²H₃] ABA, extracts wereconcentrated, and acidic-ethyl acetate soluble fractions were obtainedby a standard solvent fractionation. These fractions were purified usingBond Elut cartridge (C₁₈ and DEA, Varian) by following the methoddescribed in Wijayanti, L. et al., Biosci. Biotech. Biochem., 59:1533–1535, 1995. Samples purified from undehydrated plants were analyzedusing Senshu Pak ODS-2101-N column (100 mm length, 6 mm i. d.) (SenshuScientific Co.) by HPLC. Analytic conditions were the same as describedin Wijayanti, L. et al., Biosci. Biotech. Biochem., 59: 1533–1535, 1995.Purified samples were methylated by etherial diazomethane and analyzedby GC-SIM.

As a result, the ABA amount increased in the sense plant compared withits wild type and decreased in the antisense plant (FIG. 14).

EXAMPLE 16 Results of Evaluation of Drought Tolerance

The seeds of the obtained transformant plants were sown on an agar platesupplemented with nutritive salts (Valvekens, D. et al., Proc. Natl.Acad. Sci. USA, 85: 5536–5540, 1988), and grown under the above growingcondition for two weeks to subject to the following experiment.

Four individuals of the above plants were transplanted to plastic potswith a diameter of 9 cm filled with the soil (vermiculite:perlite=1:1)and grown under the condition with temperature of 22° C. and aphotoperiod of 16 hours. Three weeks after sowing seeds (two weeks afterthe transplantation), the pots with the plants were dehydrated bystopping watering to naturally give drought stress. Fourteen days and 17days after the initiation of non-irrigation, the pictures of the plantswere taken. The plants in which the AtNCED3 gene was introduced in theantisense direction wilted 14 days after the initiation ofnon-irrigation (FIG. 15). In contrast, the transformant plants in whichthe gene was introduced in the sense direction seldom wilted. The wildlines also wilted 17 days after the initiation of non-irrigation, whilethe transformant plants in which the gene was introduced in the sensedirection showed significant tolerance to drought (FIG. 16).

1. An isolated DNA encoding a protein having neoxanthin cleavageactivity, wherein said protein is selected from the group consisting of:(a) a protein comprising the amino acid sequence of SEQ ID NO:6; (b) aprotein encoded by a polynucleotide that hybridizes under highlystringent conditions with a DNA comprising a nucleotide sequence of SEQID NO:5, wherein said highly stringent conditions are: hybridization ina solution containing 30% formamide, 6×SSC, 5× Denhardt's solution, and100 μg/ml denatured salmon sperm DNA at 37° C. and washing in 0.1×SSC,0.1% SDS at 60° C. for 15 min; and (c) a protein comprising an aminoacid sequence that is at least 95% identical to the sequence set forthin SEQ ID NO:6.
 2. A transformed plant cell comprising the isolated DNAof claim
 1. 3. A transgenic plant comprising the transformed plant cellof claim
 2. 4. A transgenic plant which is an offspring or a clone ofthe transgenic plant of claim 3, wherein plant cells from said offspringor clone also contain the isolated DNA which encodes a protein havingneoxanthin cleavage activity and said protein is selected from the groupconsisting of: (a) a protein comprising the amino acid sequence of SEQID NO:6; (b) a protein encoded by a polynucleotide that hybridizes underhighly stringent conditions with a DNA comprising a nucleotide sequenceof SEQ ID NO:5, wherein said highly stringent conditions are:hybridization in a solution containing 30% formamide, 6×SSC, 5×Denhardt's solution, and 100 μg/ml denatured salmon sperm DNA at 37° C.and washing in 0.1×SSC, 0.1% SDS at 60° C. for 15 min; and (c) a proteincomprising an amino acid sequence that is at least 95% identical to thesequence set forth in SEQ ID NO:6.
 5. The transgenic plant of claim 3,wherein the expression level of the isolated DNA encoding a proteinhaving neoxanthin cleavage activity is increased compared to theexpression level in the wild type of said transgenic plant.
 6. Thetransgenic plant of claim 3, wherein the amount of abscisic acid isincreased compared to the wild type of said transgenic plant.
 7. Thetransgenic plant of claim 3, wherein tolerance to a stress selected fromthe group consisting of drought, high salt and low temperature stressesis increased compared to the wild type of said transgenic plant.
 8. Apropagation material comprising the isolated DNA of claim
 1. 9. A vectorcomprising the DNA of claim
 1. 10. A method for producing the transgenicplant comprising the isolated DNA of claim 1, comprising the steps ofintroducing said isolated DNA into a plant cell and regenerating a plantfrom the plant cell.
 11. A method for increasing tolerance to a stressselected from the group consisting of drought, high salt and lowtemperature stresses in a plant, wherein said method comprises: (a)introducing an isolated DNA encoding a protein having neoxanthincleavage activity into a plant cell obtained from said plant; (b)expressing the isolated DNA in said plant cell; and (c) producing aplant from the plant cell that has increased stress tolerance.
 12. Theisolated DNA of claim 1, wherein said isolated DNA encodes a proteinthat is at least 99% identical to the sequence set forth in SEQ ID NO:6.13. The isolated DNA of claim 1, wherein said isolated DNA encodes aprotein that is identical to the sequence set forth in SEQ ID NO:6. 14.The method according to claim 11, wherein the isolated DNA encodes aprotein having neoxanthin cleavage activity and said protein is selectedfrom the group consisting of: (a) a protein comprising the amino acidsequence of SEQ ID NO:6; (b) a protein encoded by a polynucleotide thathybridizes under highly stringent conditions with a DNA comprising anucleotide sequence of SEQ ID NO:5, wherein said highly stringentconditions are: hybridization in a solution containing 30% formamide,6×SSC, 5× Denhardt's solution, and 100 μg/ml denatured salmon sperm DNAat 37° C. and washing in 0.1×SSC, 0.1% SDS at 60° C. for 15 min; and (c)a protein comprising an amino acid sequence that is at least 95%identical to the sequence set forth in SEQ ID NO:6.
 15. The methodaccording to claim 11, wherein said stress is drought stress.